Plasma-based films and methods for making and using the same

ABSTRACT

The present invention relates to plasma-based films and in particular to flexible plasma-based films. The invention further relates to and to methods of making and using the flexible plasma-based films. Embodiments of the invention have been particularly developed for making flexible plasma-based films useful as a hemostat in the treatment and/or prevention of mild to severe as well as arterial bleedings, as an anti-adhesive sheet to reduce or prevent development of surgery-induced adhesions, as a wound healing patch, as a wound dressing, or as a film useful in hernia repair. Embodiments of the invention will be described hereinafter with reference to these applications. However, it will be appreciated that the invention is not limited to this particular field of use.

FIELD OF THE INVENTION

The present invention relates to plasma-based films and in particular toflexible plasma-based films. The invention further relates to methods ofmaking and using the flexible plasma-based films.

Embodiments of the invention have been particularly developed for makingflexible plasma-based films useful as a hemostat in the treatment and/orprevention of mild to severe as well as arterial bleedings, as ananti-adhesive sheet to reduce or prevent development of surgery-inducedadhesions, as a wound healing patch, as a wound dressing, or as a filmuseful in hernia repair. Embodiments of the invention will be describedhereinafter with reference to these applications. However, it will beappreciated that the invention is not limited to this particular fieldof use.

BACKGROUND OF THE INVENTION

Any discussion of the background art throughout the specification shouldin no way be considered as an admission that such art is widely known orforms part of common general knowledge in the field.

Films or sheets for use as medical products often contain embeddedmaterial such as fibers or fabrics to alter/improve the mechanicalproperties of such films or sheets, in particular to improve theirresistance to pressure. However, depending on the kind of fibers and/orfabrics used, such enforced films or sheets are known to triggercomplications in situ due to only partial biodegradability of the fibersand fabrics. Typical complications arising include the development ofconnective tissue between otherwise unrelated tissues and/or organsleading to post-surgical/surgery-induced adhesions

Post-surgical/surgery-induced adhesions can lead to severe clinicalcomplications such as loss of sensation, infertility, intestinalblockage and pelvic pain. The severity of the symptoms depends on thelocation and size of the adhesion. The number of patients suffering frompost-surgical/surgery-induced adhesions is increasing and 55%-90% ofpatients that undergo gynecological and/or abdominal surgeries facecomplications arising from adhesions.

Entirely biodegradable fibrin films are known and plasma-based filmshave been described, for example see EP 0485210 A2 and U.S. Pat. No.8,529,959 B2. As already indicated above, plasma-based, topical orimplantable medical products such as films or sheets generally have abiocompatibility advantage in comparison to similar films or sheetscomprising non-plasma based materials such as fibers and/or fabrics. Inaddition to the complications described above, plasma-based productssuch as films are suitable to provide individual patients with animplantable, plasma-based product generated from their own plasmaproviding a very high degree of biocompatibility.

EP 0485210 A2 discloses a manufacturing method for blood plasma-basedfilms comprising the clotting of 50 ml citrated plasma with 8-10 NIHunits of thrombin in a mold apparatus. However, the blood plasma-basedfilms prepared in accordance with the disclosure of EP 0485210 A2 arebrittle, and therefore inflexible, and display only poor mechanicalstrength such that the films break upon folding, making them unsuitablefor applications where flexibility and/or pressure resistance isrequired.

U.S. Pat. No. 8,529,959 B2 discloses sheets comprising a bloodplasma-derived plastic. The blood plasma-derived plastic is described asbeing obtained by clotting of blood with calcium, drying the obtainedclot and grinding of the dried clot to produce plasma clot powder, whichis mixed with glycerol to prepare a dough comprising 65% plasma clotpowder and 35% glycerol, and wherein the dough is subsequentlypressure-molded at an elevated temperature and high pressure to form thesheets. The sheets prepared in accordance with the disclosure of U.S.Pat. No. 8,529,959 B2 have a very limited flexibility and poormechanical strength such that the sheets break upon folding making themunsuitable for applications where flexibility and or pressure resistanceis required.

Accordingly, a need for improved biodegradable, flexible plasma-basedfilms or sheets exist in the field. In particular, plasma-based films orsheets having a high degree of flexibility as well as high mechanicalstrength are needed.

SUMMARY OF THE INVENTION

It is an object of the present invention to overcome or ameliorate atleast one of the disadvantages of the prior art, or to provide a usefulalternative. In particular, it is an object of the present invention toprovide improved flexible, plasma-based films and to provide improvedmethods for making and using such films, for example in the treatmentand/or prevention of medical indications.

The present invention is inter alia based on the surprising finding thatthe flexibility and/or elasticity and/or mechanical strength ofplasma-based films, when expressed as a function of fold-number, foldendurance and/or as a function of a plasma-based film's resistance toburst pressure, can be improved by mixing blood plasma with thrombinand/or calcium in excess of the amount required to induce clotting ofthe plasma, wherein the plasma is contained within a mold of a desiredvolume and shape.

Accordingly, in a first aspect, the present invention relates to amethod of making a flexible plasma-based film comprising the steps of:

-   -   (a) mixing blood plasma with more than 2 International Units        (IU) of thrombin per milliliter (ml) of the plasma and/or with        about 0.65 to 1.3 mg of calcium ions per milliliter (ml) of the        plasma to induce clotting of the plasma, wherein the plasma is        contained within a mold; and    -   (b) maintaining the plasma in the mold for the time required for        the plasma to clot and to form the film, wherein optionally        during or at the end of the time a pressure is applied to the        plasma in the mold.

Furthermore, it was surprisingly found that the flexibility ofplasma-based films can be improved by mixing blood plasma with at leastone activator of the coagulation system in a mold, and applying pressureto the plasma in the mold during or at the end of the time required forthe plasma to clot.

Accordingly, in a second aspect, the present invention relates to amethod of making a flexible plasma-based film comprising the steps of:

-   -   (a) mixing blood plasma with an activator of the coagulation        system to induce clotting of the plasma, wherein the plasma is        contained within a mold; and    -   (b) maintaining the plasma in the mold for the time required for        the plasma to clot and to form the film, wherein during or at        the end of the time a pressure ranging from about 0.3 to 125        pounds per square inch (psi) is applied to the plasma in the        mold to form the film.

In light of the above, it will be understood that the present invention,in one or more preferred embodiments, also relates to a method of makinga flexible plasma-based film comprising the steps of:

-   -   (a) mixing blood plasma with about more than 2 International        Units (IU) of thrombin per milliliter (ml) of the plasma and/or        with about 0.65 to 1.3 mg of calcium ions per milliliter (ml) of        the plasma to induce clotting of the plasma, wherein the plasma        is contained within a mold; and    -   (b) maintaining the plasma in the mold for the time required for        the plasma to clot and to form the film, wherein during or at        the end of the time a pressure ranging from about 0.3 to 125        pounds per square inch (psi) is applied to the plasma in the        mold.

In the methods of the first and second aspects, as well as inembodiments where the specific method steps of the two first aspects arecombined, the blood plasma is mixed with about 2.9 to 3.1 IU of thrombinper ml plasma to induce clotting in the mold and is subsequentlymaintained in the mold for the time required for the plasma to clot andto form the film, and a pressure ranging from about 40 to 45 psi isapplied to the plasma in the mold for about 50 to 70 seconds during orat the end of the time required for the plasma to clot.

In a third aspect, the present invention relates to a flexibleplasma-based film when prepared by the methods according to the first orsecond aspects.

In a fourth aspect, the present invention relates to a flexibleplasma-based film comprising between 0.1 to 10 IU of thrombin per ml ofplasma and having a thickness ranging from about 0.005 to 0.1 mm,wherein the flexible film is characterized by a fold number of at least1, such as at least 2, such as at least 4, such as at least 5.Preferably, in the embodiments of the present invention the flexibleplasma-based films can also be characterized: by a fold endurance of atleast 10, such as at least 20, such as at least 30, such as at least 40,such as at least 50, such as at least 60, such as at least 70, such isat least 80, such as at least 90, such is at least 100; and/or by aburst pressure of about 50 to 1000 mm Hg, or of about 100 to 1000 mm Hg,or of about 100 to 800 mm Hg, or of about 100 to 600 mm Hg, or of about100 to 500 mm Hg, or of about 100 to 450 mm Hg, or of about 140 mm Hg,or of about 150 mm Hg, or of about 175 mm Hg, or of about 200 mm Hg, orof about 225 mm Hg, or of about 250 mm Hg, or of about 275 mm Hg, or ofabout 300 mm Hg, or of about 325 mm Hg, or of about 350 mm Hg, or ofabout 375 mm Hg, or of about 400 mm Hg.

In a fifth aspect, the present invention relates to the flexibleplasma-based films according to the third or fourth aspects for use as ahemostat, preferably as a hemostat to stop a mild to severe bleeding.Accordingly, in embodiments of the fifth aspect, the invention alsorelates to a method of stopping a mild to severe bleeding, comprisingapplying the flexible plasma-based film according to the third or fourthaspect as a hemostat.

In a sixth aspect, the present invention relates to the flexibleplasma-based films according to the third or fourth aspects for use as ahemostat to stop an arterial bleeding. Accordingly, in embodiments ofthe sixth aspect, the invention also relates to a method of stopping anarterial bleeding, comprising applying the flexible plasma-based filmaccording to the third or fourth aspect as a hemostat.

In a seventh aspect, the present invention relates to the flexibleplasma-based films according to the third or fourth aspects for use asan anti-adhesive sheet to reduce or prevent development of asurgery-induced adhesion. Accordingly, in embodiments of the seventhaspect, the invention also relates to a method of reducing or preventingdevelopment of surgery-induced adhesions, comprising applying theflexible plasma-based film according to the third or fourth aspect as ananti-adhesive sheet.

In an eighth aspect, the present invention relates to the flexibleplasma-based films according to the third or fourth aspects for use as awound healing patch. Accordingly, in embodiments of the eighth aspect,the invention also relates to a method of treating a wound, comprisingapplying the flexible plasma-based film according to the third or fourthaspect as a wound healing patch.

In a ninth aspect, the present invention relates to the flexibleplasma-based films according to the third or fourth aspects for use as awound dressing. Accordingly, in embodiments of the ninth aspect, theinvention also relates to a method of treating a wound, comprisingapplying the flexible plasma-based film according to the third or fourthaspect as a wound dressing.

In a tenth aspect, the present invention relates to the flexibleplasma-based films according to the third or fourth aspects for use inhernia repair. Accordingly, in embodiments of the tenth aspect, theinvention also relates to a method of treating a hernia, comprisingapplying the flexible plasma-based film according to the third or fourthaspect.

FIGURES

Embodiments of the invention will now be described, by way of exampleonly, with reference to the accompanying drawings in which:

FIG. 1 schematically shows a flexible plasma-based film (1) according toan embodiment of the present invention.

FIG. 2 schematically illustrates a plasma-based film (1) like the oneshown in FIG. 1 comprising one or more pharmaceutically-active agents(2).

FIG. 3 schematically illustrates a plasma-based film (1) like the oneshown in FIG. 1 comprising one or more pharmaceutically-active agents(2) embedded throughout the film to which two furtherpharmaceutically-active agents (3, 4) have been applied by subsequentspray-drying. In some embodiments the two furtherpharmaceutically-active agents may be spray-dried plasma (3) and aspray-dried activator of the coagulation system (4).

FIG. 4 schematically illustrates a vertical section through an apparatussuitable for making the plasma-based film of the present invention,where (5) is a downward-force piston, (6) is a non-porous plate forevenly applying pressure to the clotting/clotted plasma (not shown) inthe mold (7) and comprising a liquid removal system of absorbentmaterial (8) and vacuum-driven suction (9).

DETAILED DESCRIPTION OF THE INVENTION

In order to provide a clear and consistent understanding of thespecification and claims, and of the scope to be given such terms, thefollowing definitions are provided.

Definitions

In the context of the present application, the term “flexible” meansthat a structure such as a plasma-based film is capable of bending, evenin tight turns, without breaking. In particular, a flexible film in thecontext of the present application is capable of folding withoutbreaking and, as such, the film's degree of flexibility can be expressedby way of fold numbers or fold endurance as defined below. Further, afilm's degree of flexibility can be expressed by the film's burstpressure also as defined below.

In the context of the present application, the term “plasma based”, ingeneral, means structures and materials such as the films according tothe present invention, and in the case of multi-layered films theindividual film layers, which are produced from blood plasma as thesource material. In particular, if the majority of the components ofsuch structures or materials is plasma, they are understood to beplasma-based structures or materials.

In the context of the present application, the term “film”, in generalmeans, thin, substantially planar, membranous structures, which can alsobe described as sheets. Notwithstanding, the term film in the context ofthe present application expressly encompasses single-layered films orsheets as well as composite multi-layered films or sheets. Further, afilm in the context of the present application may comprisepharmaceutically-active agents either topically applied to the film,homogeneously distributed throughout the film or encapsulated betweenthe film layers of a multi-layered film. The term “film” alsoencompasses structures having a degree of porosity that allows an uptakeand/or release of substances such as pharmaceutically-active agents fromthe film. As such, the term “film” in the context of the presentapplication also encompasses a thin, substantially planar, membranousscaffold upon which, or within which, pharmaceutically-active agents maybe contained. Accordingly, the term film when used to describeembodiments of the present invention also encompasses plasma-basedvessels or delivery devices for pharmaceutically-active agents comprisedon or within the film.

In the context of the present application, the term“pharmaceutically-active agent” means a substance, particle and/or drugused to furnish pharmacological activity or to otherwise have a directeffect in the diagnosis, cure, mitigation, treatment or prevention of amedical indication, or to have a direct effect in restoring, correctingor modifying physiological functions.

In the context of the present application, the term “agent increasingthe degree of cross linking between fibrin polymers” means agents thatincrease the number of links between the y- and a-chains of fibrinmolecules thereby stabilizing fibrin fibers. Such agents, amongstothers, include activated coagulation factor XIII (FXIIIa), calciumchloride, modified fibrin molecules such as mutated truncated fibrinmolecules, and chain-specific antibodies.

In the context of the present application, the term “fold number” meansthe number of times a structure such as a plasma-based film can be bentin tight turns until the film disrupts. A simple test of determiningfold number is described below.

In the context of the present application, the term “fold endurance”means the number of times a structure such as a plasma-based film canrepeatedly be folded in a tight turn and again unfolded to its originalposition. Fold endurance in the context of the present applicationprovides a means for wear resistance estimation of a flexible structure.

In the context of the present application, the term “burst pressure”means the maximum pressure a structure can withstand before breaking ordisrupting. In the context of the present application the term burstpressure particularly means the maximum pressure a plasma-based film canwithstand before breaking or disrupting.

In the context of the present application, the term “time required forplasma to clot” is given its plain-English meaning, i.e. it means thetime required for the plasma used in the preparation of the plasma-basedfilms described herein to clot.

In the context of the present application, the term “activator of thecoagulation system” means substances, particles or agents that initiatecoagulation/clotting. Such activators may initiate coagulation at anystage of the coagulation cascade and, as such, include agents acting onthe intrinsic, extrinsic and common pathways of coagulation.

In the context of the present application, the term “hemostat” meansdevices suitable to reduce and ultimately stop bleeding. In particular,the term encompasses plasma-based films suitable to close blood vessels.As such, hemostats in the context of the present application includeplasma-based films that can close blood vessels as a physical barrier,plasma-based films that can deliver hemostatic agents to the site ofbleeding to close the blood vessel as well as combinations thereof.

In addition to the above definitions, and unless the context clearlyrequires otherwise, throughout the description and the claims, the words“comprise”, “comprising”, and the like are to be construed in aninclusive sense as opposed to an exclusive or exhaustive sense; that isto say, in the sense of “including, but not limited to”.

Further, reference throughout this specification to “one embodiment”,“some embodiments” or “an embodiment” means that a particular feature,structure or characteristic described in connection with the embodimentis included in at least one embodiment of the present invention. Thus,appearances of the phrases “in one embodiment”, “in some embodiments” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment, but may.Furthermore, the particular features, structures or characteristics maybe combined in any suitable manner, as would be apparent to one ofordinary skill in the art from this disclosure, in one or moreembodiments.

As used herein, unless otherwise specified the use of the ordinaladjectives “first”, “second”, “third”, etc., to describe a commonobject, merely indicate that different instances of like objects arebeing referred to, and are not intended to imply that the objects sodescribed must be in a given sequence, either temporally, spatially, inranking, or in any other manner.

As used herein, the term “exemplary” is used in the sense of providingexamples, as opposed to indicating quality. That is, an “exemplaryembodiment” is an embodiment provided as an example, as opposed tonecessarily being an embodiment of exemplary quality.

Flexible, Plasma-Based Films of the Invention and Methods for Making andUsing the Same

As described above, the flexibility, elasticity and/or mechanicalstrength of plasma-based films is important for such plasma-based filmsto be efficiently and successfully used as hemostats for stopping mildto severe bleedings and even for stopping arterial bleedings. Inparticular, the plasma-based films of the present invention provide therequired flexibility, elasticity and mechanical strength required forthe films to seal such bleeds, i.e. the flexibility necessary for thefilm to conform to the physiological topology of the site of the bleedand to withstand even arterial blood pressure at the site of the bleed.

Similarly, the flexibility, elasticity and mechanical strength ofplasma-based films according to the present invention is important forsuch plasma-based films to be efficiently used as anti-adhesive sheetsto reduce or prevent the development of surgery-induced adhesions.

Post-surgical/surgery-induced adhesions result from the formation ofscar tissue following surgery. While adhesion is part of the normalhealing response to tissue trauma, it can lead to severe post-surgicalcomplications when a stable cross-connection between otherwiseunconnected tissues arises as a result of adhesions.

In fact, more than 90% of patients develop peritoneal adhesionsfollowing abdominal or pelvic surgery. In most patients the adhesivetissue is subsequently remodeled and ultimately dissolved. However, inmillions of patients adhesions form between organs and/or tissues thatare normally separate, such as the intestines, bowels, uterus, ovariesand other organs, causing dysfunction and other possibly severecomplications. For example, within the abdominal cavity adhesions canlead to small bowel obstruction, female infertility and pain. Inaddition, adhesions often make subsequent surgery more difficult andlimit subsequent treatment options. For example, adhesions often impedesubsequent minimally invasive surgery.

The plasma-based films of the present invention are suitable to provideanti-adhesion barrier sheets designed to act as a physical shieldkeeping damaged tissue separate from the surrounding tissue, therebypreventing undesirable adhesion. The plasma-based film when used as ananti-adhesive sheet separates the traumatized tissue during its initialtime of healing but will ultimately break down entirely due to itsbiodegradation. In this respect, it is noteworthy that adhesion growthinitiates within the first 3-5 days after surgery. As such, theplasma-based films of the present invention are particularly useful asanti-adhesive sheets allowing the separation of traumatized tissue fromsurrounding tissue during these first days of post-surgical healing,thereby reducing or preventing the development of surgery-inducedadhesions and, ultimately, reducing or preventing at the risk ofcomplications arising due to the formation of surgery-induced adhesions.As will be appreciated, the flexibility of the plasma-based films of thepresent invention provides a significant advantage when such films areused as anti-adhesive sheets due to their ability to conform to thephysiological topology of the traumatized tissue thereby providing anefficient anti-adhesive barrier between the traumatized tissue andsurrounding tissues.

Due to their biocompatibility and complete biodegradation over time, theflexible plasma-based films such as the film (1) schematically shown inFIG. 1 are also particularly suitable for use as wound dressings, woundhealing patches, or for use in the repair of hernias or vascularlesions. In this regard, it is noteworthy that the plasma-based films ofthe present invention are typically non-toxic, non-interfering withnormal healing, non-interfering with immunological functions, applicablein various settings such as bleeding, infections and anastomotic surgeryand are, due to their flexibility, elasticity and high mechanicalstrength easy to handle and easy to apply.

Further, the plasma-based films of the present invention can serve ascarriers, vessels and/or delivery devices for pharmaceutically-activeagents, for example for agents useful in the treatment of theabove-mentioned medical applications. For example, if the film is usedas a wound healing patch, the film may carry one or morepharmaceutically-active wound healing agents, i.e. agents supportingwound healing. Alternatively, the films of the present invention may beused as bare wound dressings.

As such, the plasma-based films of the present invention such as thefilm (1) schematically illustrated in FIG. 2 may comprise at least onepharmaceutically-active agent (2). Preferably, the at least onepharmaceutically-active agent is selected from the group consisting of:antibiotic agents; anti-inflammatory agents; anti-infective agents;growth factors; chemokines; immunomodulators; wound healing agents;activators of the coagulation system; anti-coagulation agents;anti-adhesion agents; anti-fibrinolytic agents; penicillin; silver;chlorhexidine; cells; stromal cell-derived chemokines; stromalcell-derived factor 1 alpha; stromal cell-derived factor 1 beta;fibrinogen; Factor Vila; CXCL-12; heparin; aprotinin; tranexamic acid;non-ionic surfactants; Pluronic F68; TWEEN 80; COX-2 inhibitors; andNimesulide.

The films of the present invention may be specifically engineered asdelivery devices for the above-listed pharmaceutically-active agents.For example, a need exists for topically-applicable or implantableflexible plasma-based films, which may contain thepharmaceutically-active agents and/or deliver them to a treatment site.

Non-ionic surfactants, such as Polyoxyethylene sorbitan esters, inparticular Polyoxyethylene (20) sorbitan monooleate (TWEEN-80®),Polyoxyethylene-polyoxypropylene block copolymers, in particularPluronic F68®, or COX-2 inhibitors, in particularN-(4-Nitro-2-phenoxyphenyl)methanesulfonamide (Nimesulide®), orcombinations thereof are particularly useful when plasma-based films foruse as anti-adhesive sheets to reduce or prevent the development ofsurgery-induced adhesions are to be prepared. In such films, theseagents may homogeneously be distributed throughout the film, or may beapplied topically to the film, either alone or as a combination ofagents.

In some embodiments the agent is homogenously distributed throughout thefilm. In other embodiments the agent is topically applied to the film.In yet further embodiments of the present invention, the plasma-basedfilm may comprise a pharmaceutically-active agent homogenouslydistributed throughout the film and further comprise the same or anotherpharmaceutically-active agent topically applied to a surface of thefilm.

In embodiments where the agents are homogeneously distributed throughoutthe film, the agents become “trapped” within the film during formationof the plasma-based films due to clotting of the plasma. Afterimplantation the plasma-based film will degrade (mainly due to lyticfactors, hydration and mechanical wear) thereby successively releasingthe pharmaceutically-active agents distributed throughout.

When an agent is topically applied to a film according to the invention,the agent can be applied by spray-coating, spray-drying, brushing and/orsoaking.

Stromal cell derived chemokines are particularity interesting for usewith films of the present invention. Stromal cell-derived factors1-alpha and 1-beta are small cytokines that belong to the chemokinefamily, members of which activate leukocytes and are often induced inresponse to pro-inflammatory stimuli such as lipopolysaccharides, tumornecrosis factor (TNF) or interleukin-1 (IL1). Chemokines such as CXCL-12inhibit the formation of connective tissue and are therefore usefulagents for application to or distribution throughout films of thepresent invention when used as anti-adhesive sheets.

Once the films have been prepared, agents that inhibit further fibrinformation, such as heparin, aprotinin, tranexamic acid or otheranti-fibrinolytics can also be added to plasma-based films to be used asanti-adhesive sheets, for example by topical application.

In some embodiments, the agent can be a single agent or can be a mixtureof agents such as proteins, for example comprising a mixture ofactivators of the coagulation system such as plasma itself.

In some embodiments, the plasma-based films of the present invention maycomprise more than one pharmaceutically-active agent homogenouslydistributed throughout the film and/or more than onepharmaceutically-active agent topically applied to the film.

In one or more preferred embodiments

-   -   (i) a first pharmaceutically-active agent is topically applied        to the film, optionally by spray-drying,    -   (ii) a separation layer is subsequently applied onto the        topically-applied first agent, and    -   (iii) a second pharmaceutically-active agent is topically        applied onto the separation layer.

In such embodiments the first pharmaceutically-active agent may befibrinogen, the separation layer may comprise starch, optionallygelatinized starch, and the second pharmaceutically-active agent may bethrombin. Preferably the fibrinogen is applied by spray-drying on a filmlayer, application of still-liquid gelatinized starch (prepared forexample by gelatinizing 2.5 g of starch in 50 ml water by heating to125° C.) on top of the fibrinogen layer and applying a pressure or about15.5 psi (105 kPa) to the film. Typically, 2 ml of gelatinized starchare sufficient to cover a 4″×4″ film with a separation layer.Subsequently, thrombin is applied onto the separation layer.Spray-drying of about 5 ml of a 300 IU/ml thrombin solution on a 4″×4″film proved to be most effective.

In alternative embodiments, the film of the present invention maycomprise a topically applied pharmaceutically-active agent for use as ahemostat on just one outer surface of the layered film, while one of theinner layers comprises a different agent, for instance an antibiotic.The topically applied hemostatic agent on the film surface may bereleased and act immediately upon application of the film to a woundthereby providing an immediate beneficial hemostatic effect, while theantibiotic agent from the inner layer(s) of the film is only releasedafter the immediate hemostatic effect, thereby providing an antibioticeffect during subsequent healing stages. As such, in such embodiments,topical application can provide for an immediate release effect of anagent, whereas homogeneous distribution throughout the film may allowfor a retarded-release or slow-release effect of an agent.

In other embodiments, and referring to FIG. 3, the plasma-based film (1)optionally comprising a pharmaceutically active agent (2) homogeneouslydistributed throughout, can also be coated with spray-dried plasma (3)to provide a layer of plasma proteins to the outer surface of the film.Air gun spraying can be utilized for topical application of plasma as itmaintains the plasma proteins' activity after drying. In effect thistopical application by spray-drying provides a plasma-based filmcomprising active coagulation proteins on its outer surface. Theactivities of fibrinogen and FXa of the clotting system remain intactsuch that the film can be used as a hemostatic dressing with a spraydried layer of active coagulation proteins. In addition, a final layerof an activator of the coagulation system (4) such as a thrombinconverter can be sprayed on top of the spray-dried plasma. For example,the thrombin converter may be selected from: tissue factor, recombinanttissue factor, thrombin, kaolin, diatomaceous earth, and activators ofthe coagulation system. The so modified plasma-based film can be used asa hemostat for mild, moderate or severe bleedings.

Further, a flexible plasma-based film according to the present inventionmay be a composite film comprising several plasma-based film layers,i.e. a multi-layered plasma-based film. In some multi-layeredplasma-based films each individual film layer may comprise no, the sameor different pharmaceutically-active agents.

The degree of cross-linking between fibrin polymers of the flexibleplasma-based film influences the degradation speed of the film, whereina high degree of cross-linking correlates with slow degradation, andwherein a low degree of cross-linking correlates with fast degradation.Accordingly, controlling the degree of cross-linking allows for thecontrol of the rate or speed of degradation and therefore allows for thefilm to be customized according to its application.

For example, if the film is to be used as an anti-adhesive sheet, thefilm should degrade within one to two weeks of implantation. Incontrast, films used for hernia repair should have a much slowerdegradation speed leading to an almost-permanent implant, preferablyrequiring several weeks to months or, in particular instances, evenseveral months to years, before complete degradation of the film. Ingeneral, the degradation speed can be optimized to suit a film'sparticular function, i.e. to promote healing, provide mechanicalstability for a rupture or lesion covered by the film, etc.

Similarly, the degree of cross-linking between fibrin polymers of thefilm influences the release time of a pharmaceutically-active agenthomogeneously distributed within the film, wherein a high degree ofcross-linking correlates with longer release times, and wherein a lowdegree of cross-linking correlates with shorter release times.Accordingly, controlling the degree of cross-linking allows for thecontrol of the release rate of a pharmaceutically-active agenthomogeneously distributed throughout the films of the present invention.

The combination of the different features of the films described herein,i.e. monolayer films, multilayer films, different agents either appliedtopically or distributed throughout the film, allow for the productionof films tailor-made for specific applications and even individualpatients.

The flexible plasma-based film of the present invention is typicallyprepared from liquid plasma, where the plasma may be human or animalplasma. For example, the plasma is Fresh Frozen Plasma (FFP) or ispathogen-safe plasma. FFP can be obtained from a blood bank. However,plasmas obtained from a professional plasma manufacturer such as CLSBehring, Baxter or Octapharma are typically preferred as they providemuch higher pathogen safety due to pathogen removal and inactivationmethods incorporated in their production processes. Plasmas from plasmamanufacturers are usually pooled and pathogen-safe plasmas.

In embodiments where pathogen-safe plasma is used as the source materialfor the plasma-based films of the present invention, the plasma haspreferably been subjected to viral inactivation treatment, has beenpasteurized, has been radiated, and/or has been nano-filtered.Typically, the pathogen-safe plasma being used as source material forthe films of the present invention has been subjected tosolvent/detergent treatment (S/D treatment) to inactivate any viralpathogens possibly contained therein.

In one or more preferred embodiments, the plasma-based films of thepresent invention have a thickness ranging from about 0.005 to 0.1 mm,or from about 0.005 to 0.09 mm, or from about 0.0075 to 0.08 mm, or fromabout 0.01 to 0.08 mm, or from about 0.01 to 0.07 mm, or from about0.015 to 0.065 mm, or from about 0.02 to 0.06 mm, or from about 0.02 to0.055 mm, or from about 0.02 to 0.05 mm, or from about 0.02 to 0.04 mm,or from about 0.02 to 0.03 mm, or about 0.01 mm, or about 0.02 mm, orabout 0.03 mm, or about 0.04 mm, or about 0.05 mm, or about 0.06 mm.

As already indicated above, the particularly advantageous flexibility,elasticity and mechanical strength of the flexible plasma-based films ofthe present invention can be expressed by way of fold numbers, foldendurance and burst pressure of the films.

A simple test to determine film flexibility is the evaluation of afilm's fold number. Such determination is accomplished by folding onehalf of the film on top of the other half, turning the stack by 90° andagain folding one half of the stack on top of the other and so on untilthe film breaks or rupture. Each folding without triggering film ruptureincreases the fold number by 1. This test is particularly useful for theassessment of a plasma-based film's ability to maintain its integritywhen being bent in tight turns. The flexible plasma-based films of thepresent invention are typically characterized by a fold number of atleast 1, such as at least 2, such as at least 3, such as at least 4,such as at least 5.

Another test to determine film flexibility is the evaluation of foldendurance. Such determination is accomplished by repeatedly folding onehalf of the film on top of the other half and unfolding to its originalposition. Fold endurance is expressed by the number of suchfolding/unfolding repeats and provides a means for wear resistanceestimation. The flexible plasma-based films of the present invention aretypically characterized by a fold endurance of at least 10, such as atleast 20, such as at least 30, such as at least 40, such as at least 50,such as at least 60, such as at least 70, such is at least 80, such asat least 90, such as at least 100.

Still another method for determination of film flexibility is thedetermination of burst pressure according to the Standard Test Methodfor Burst Strength of Surgical Sealants (ASTM-F 2392-04). A film isfastened in a fixture according to ASTM-F 2392-04 and the fixture isconnected to a pump with a pressure transducer being attached inlinebetween the pump and the burst pressure fixture. Pumping a fluid intothe system increases the pressure until the film bursts. Thus obtainedburst pressure is indicated as [mm Hg]. The flexible plasma-based filmsof the present invention are typically characterized by a burst pressureof about 50 to 1000 mm Hg, or of about 100 to 1000 mm Hg, or of about100 to 800 mm Hg, or of about 100 to 600 mm Hg, or of about 100 to 500mm Hg, or of about 100 to 450 mm Hg, or of about 140 mm Hg, or of about150 mm Hg, or of about 175 mm Hg, or of about 200 mm Hg, or of about 225mm Hg, or of about 250 mm Hg, or of about 275 mm Hg, or of about 300 mmHg, or of about 325 mm Hg, or of about 350 mm Hg, or of about 375 mm Hg,or of about 400 mm Hg.

In some preferred embodiments of the present invention a flexibleplasma-based film characterized by a burst pressure of at least 130 to240 mm Hg, when the dry film (with about 4% residual water) has a filmthickness of 0.03 mm, in particular by a burst pressure of 170 to 240 mmHg for a 0.03 mm film in dry condition, or 130 to 180 mm Hg for a 0.03mm film (film thickness in dry condition) after soaking for 10 minutesin an aqueous solution containing 10% glycerol is provided.

In other preferred embodiments of the present invention a flexibleplasma-based film characterized by a burst pressure of at least 190 to450 mm Hg when the dry film (with about 4% residual water) has a filmthickness of 0.05-0.06 mm, in particular by a burst pressure of 300 to450 mm Hg for a 0.05-0.06 mm film in dry condition, or 190 to 400 mm Hgfor a 0.05-0.06 mm film (film thickness in dry condition) after soakingfor 10 minutes in an aqueous solution containing 10% glycerol isprovided.

Tensile strength was determined according to the Standard Test Methodfor Strength Properties of Tissue Adhesives in Lap-Shear by TensionLoading (ASTM F2255-05) by measurement with an Instron Model 58R4505Mechanical Test System using a 50 N (˜10 #) loadcell and a crossheadspeed of 1.0 inch per minute. The samples had dogbone shape with anarrow region of 0.25″ and were placed in the instrument with rubberlined pneumatic grips with the pressure set at 20 psi. The obtainedtensile strength is given as pound-force [lbf]. The flexibleplasma-based films of the present invention are typically characterizedby a tensile strength of at least 0.25 lbf (approximately 1.1 N), suchas at least 0.5 lbf (approximately 2.2 N), such as at least 0.75 lbf(approximately 3.3 N), such as at least 1 lbf (approximately 4.4 N),such as at least 1.2 lbf (approximately 5.28 N), or by a tensilestrength ranging from about 0.25 lbf (approximately 1.1 N) to 1.5 lbf(approximately 6.6 N), such as from about 0.5 lbf (approximately 2.2 N)to 1.5 lbf (approximately 6.6 N), such as from about 0.7 lbf(approximately 3.08 N) to 1.5 (approximately 6.6 N), such as from about0.8 lbf (approximately 3.52 N) to 1.5 lbf (approximately 6.6 N), such asfrom about 0.9 lbf (approximately 3.96 N) to 1.5 lbf (approximately 6.6N), such as from about 1 lbf (approximately 4.4 N) to 1.5 lbf(approximately 6.6 N).

In some preferred embodiments the flexible plasma-based film of thepresent invention comprises one or more humectants. In particular, thefilm may be coated with humectants such as glycerol, for instance bysoaking in 10% glycerol for 10 to 15 minutes, to prevent the film frombecoming brittle during storage. It is also possible to admix thehumectant during clotting of the plasma when preparing the film. In suchembodiments, admixing 1-2% of glycerol, based on the total weight of theclotting mixture, is typically sufficient to prevent films from becomingbrittle during storage.

Alternatively, the film may be packaged or pouched in ahumidity-controlled container after preparation to preserve the film'sflexibility. The pouch or container may be a foil pouch or anothersterilization container. Typically the plasma-based films of the presentinvention are sterilized, preferably by gamma sterilization, e-beamsterilization, and/or UV sterilization

An adhesive backing can be applied to the film to provide additionaladhesion of the film to the tissue if so desired. Notwithstanding,typically, the plasma-based films of the present invention without anadhesive backing adhere to the tissue they are applied to such that staysutures were generally not required during in situ testing in animalstudies.

As indicated above, it was surprisingly found that the flexibleplasma-based films of the present invention having the above-describedcharacteristics and features can advantageously be prepared by themethods of the first and second aspects of the present inventionmentioned above, i.e. that the flexibility, elasticity, and mechanicalstrength of plasma-based films can be improved by the particular methodsof preparation.

Namely, in the first aspect, the present invention relates to a methodof making a flexible plasma-based film comprising the steps of:

-   -   (a) mixing blood plasma with more than 2 International Units        (IU) of thrombin per milliliter (ml) of the plasma and/or with        about 0.65 to 1.3 mg of calcium ions per milliliter (ml) of the        plasma to induce clotting of the plasma, wherein the plasma is        contained within a mold; and    -   (b) maintaining the plasma in the mold for the time required for        the plasma to clot and to form the film, wherein optionally        during or at the end of the time a pressure is applied to the        plasma in the mold.

Furthermore, it was surprisingly found that the flexibility ofplasma-based films can be improved by mixing blood plasma with at leastone activator of the coagulation system in a mold, and applying pressureto the plasma in the mold during or at the end of the time required forthe plasma to clot. The mold preferably has a volume and shape suitablefor the preparation of the films of the present invention.

As such, and in accordance with the second aspect, the present inventionrelates to a method of making a flexible plasma-based film comprisingthe steps of:

-   -   (a) mixing blood plasma with an activator of the coagulation        system to induce clotting of the plasma, wherein the plasma is        contained within a mold; and    -   (b) maintaining the plasma in the mold for the time required for        the plasma to clot, wherein during or at the end of the time a        pressure ranging from about 0.3 to 125 pounds per square inch        (psi) is applied to the plasma in the mold to form the film.

In one or more preferred embodiments, the pressure ranges from about 30to 95 psi, or from 30 to 56 psi, or from 40 to 50 psi, or from 40 to 45psi, or is 44 psi. Preferably, the pressure is applied for only afraction of time required for the plasma to, such as for 30 to 120seconds, such as for 45 to 85 seconds, such as for 50 to 70 seconds,such as for 60 seconds.

Typically, the thrombin in the method of the first aspect or theactivator of the coagulation system in the method of the second aspectare present in excess, such that the time required for the plasma toclot is relatively short ranging from about 5 to 20 minutes from mixingof the thrombin or the activator of the coagulation system with theplasma, or from about 10 to 20 minutes, or from about 12 to 18 minutes.In some embodiments the time required for the plasma to clot is 15minutes. Accordingly, in some embodiments the pressure is applied afterabout 5 to 20 minutes from mixing of the thrombin of the activator ofthe coagulation system with the plasma, or after about 10 to 20 minutes,or after about 12 to 18 minutes, or after about 15 minutes.

In this regard, it is noteworthy that induction of clotting can beachieved in some instances simply by recalcification of the plasma.Alternatively, clotting can be induced by the addition of agents thatconvert the fibrinogen in the plasma to fibrin such as thrombin andthrombin-like coagulation activators. In addition, the extrinsic pathwayof the coagulation system can be activated to induce clotting of theplasma. Such extrinsic activators are phospholipids, phospholipidscontaining, phosphatidylserine and phosphatidylcholine, tissue factor,recombinant tissue factor such as Dade Innovin, diatomaceous earth, orother common extrinsic pathway activators.

Accordingly, in some embodiments of the methods of the second aspect,the activator of the coagulation system is preferably selected from oneor more members of the group consisting of: thrombin and thrombin-likecoagulation activators; calcium; fibrinogen-to-fibrin converters;phospholipids; phosphatidylcholine; tissue factor; diatomaceous earth;zeolithes; kaolin; Factor Vila; and Factor Xa. In some embodiments ofthe methods of the second aspect the activator of the coagulation systemis thrombin and/or calcium.

In some embodiments of the methods of the first or second aspects, theplasma is mixed with about 0.1 to 10 International Units (IU) ofthrombin per milliliter (ml) of the plasma and/or with about 0.65 to 1.3mg of calcium ions per milliliter (ml) of the plasma. Preferably theplasma is mixed with about 0.1 to 7 IU of thrombin per ml of plasma, orwith about 0.15 to 6 IU of thrombin per ml of plasma, or with about 1 to5 IU of thrombin per ml of plasma, or with about 2 to 4 IU of thrombinper ml of plasma, or with about 2.5 to 3.5 IU of thrombin per ml ofplasma, or with about 2.9 to 3.1 IU of thrombin per ml of plasma, or theplasma is mixed with more than 2 IU of thrombin per ml of plasma, suchas more than 2.5 IU of thrombin per ml of plasma, such as more than 2.75IU of thrombin per ml of plasma, such as more than 3 IU of thrombin perml of plasma, such as more than 3.25 IU of thrombin per ml of plasma,such as more than 3.5 IU of thrombin per ml of plasma, such as more than3.75 IU of thrombin per ml of plasma, such as more than 4 IU of thrombinper ml of plasma and/or with about 0.65 to 1.3 mg of calcium ions permilliliter (ml) of the plasma.

In some embodiments of the methods of the first or second aspects, thesteps of mixing and maintaining the plasma are performed at atemperature range from room temperature to about 40° C., or from 36 to38° C., or at 37° C.

In some further embodiments of the methods of the first or secondaspects, the pressure is applied to the plasma within the mold via aplate to form the film, preferably the plate is pressed onto the plasmain the mold by a pneumatic press.

In some embodiments, the methods of the first or second aspects furthercomprise a step of:

-   -   (c) removing excess liquid from the mold.

During this further step, the excess liquid is typically extruded fromthe clotted or clotting plasma when the pressure is applied. Preferablythe excess liquid is removed by way of a liquid removal system,optionally selected from: a suction system such as a vacuum-drivensuction system; and a system comprising adsorbent materials such asabsorbent cloths, towels, membranes or gels.

For example, and referring to FIG. 4, the pressure can be applied to theclotting mixture (5) via a solid, non-porous plate (6) being slightlysmaller than the outer perimeter of the mold (7). The plate is thenplaced under a liquid removal system (8) that will remove the excessaqueous portion of the plasma-based film during compression. Once theaqueous removal system (8) is in place force is uniformly placed on theplate (6), for example by way of a downward-force piston (9) of apneumatic press. The force on the plate pressing down on the clottingmixture in the mold can be very low, e.g. as low as 5 pounds, or theforce can be high, e.g. 2000 pounds. During application of the pressure,the excess aqueous portion of the plasma-based film, which containsprimarily blood serum and excess water, is extruded or “squished” sothat the excess aqueous portion of the plasma-based film is displaced tothe outer perimeter of the plate (6). During this displacement step theaqueous phase is removed by the liquid removal system (8) via avacuum-driven suction and can also be collected via absorbent material(10). At the end of the compression, the “squished” film can be removedfrom the container as a flat, highly compressed, low water content, highstrength film.

In some embodiments, the methods of the first or second aspects furthercomprise a step of:

-   -   (d) drying the plasma-based film.

During this further step, the film is typically dried at roomtemperature and/or under negative pressure conditions such as in alaminar flow unit or a vacuum unit, or at increased temperatures such asin an oven.

In some embodiments the film prepared further comprises at least onepharmaceutically-active agent, preferably the agent is homogeneouslydistributed throughout the film or is topically applied to the film andmay be selected from the group consisting of: antibiotic agents;anti-inflammatory agents; anti-infective agents; growth factors;chemokines; immunomodulators; wound healing agents; activators of thecoagulation system; anti-coagulation agents; anti-adhesion agents;anti-fibrinolytic agents; penicillin; silver; chlorhexidine; stromalcell-derived chemokines; stromal cell-derived factor 1 alpha; stromalcell-derived factor 1 beta; fibrinogen; Factor Vila; CXCL-12; heparin;aprotinin; tranexamic acid; non-ionic surfactants; Pluronic F68; TWEEN80; COX-2 inhibitors; and Nimesulide.

In some further embodiments of the methods of the first or secondaspects, the at least one pharmaceutically-active agent is homogeneouslydistributed throughout the film. In such embodiments, the step of mixingthe blood plasma with the thrombin and/or calcium or with the activatorof the coagulation system also comprises mixing the plasma with the atleast one pharmaceutically-active agent to provide a clotting mixturewith the proviso that the agent cannot be an anti-coagulation agent.Typically, the at least one pharmaceutically-active agent may constituteup to 5% of the total weight of the clotting mixture, such as up to 2%,preferably between 1 and 2%.

In this regard, it is noteworthy that admixture of the agents up to 5%of the total weight of the clotting mixture did not compromisemechanical stability of films produced. As shown in more detail in theExamples below, addition of 1-2% of anti-adhesive agents was sufficientfor the films to display no to very minor adhesions in an animal study.In the animal model for the development of adhesions, the anti-adhesivesheets of the present invention led to less than 25% of the initiallyapplied wound surface being covered with adhesions, in particular theydisplayed 0-15% wound surface coverage. In contrast, the best-performingcommercially-available anti-adhesion film tested in the same animalmodel for the development of adhesions led to a wound surface coverageof at least 40%.

In some further embodiments of the methods of the first or secondaspects, the at least one pharmaceutically-active agent is topicallyapplied to the film, optionally the agent can be applied byspray-coating, spray-drying, brushing and/or soaking. In some preferredembodiments, the agent can be a mixture of proteins comprisingactivators of the coagulation system such as plasma.

In some further embodiments of the methods of the first or secondaspects, the film may comprise several film layers prepared bysuccessively repeating the steps of the methods of the invention in themold to form a multi-layered plasma-based film, wherein, optionally,each individual film layer may comprise no, the same or differentpharmaceutically-active agents. Preferably, severalpharmaceutically-active agents may be topically applied simultaneouslyor successively.

In some further embodiments of the methods of the first or secondaspects, the step of mixing the blood plasma with the thrombin and/orcalcium or with the activator of the coagulation system also comprisesmixing the plasma with an agent increasing the degree of cross-linkingbetween the fibrin polymers generated during clotting of the plasma,preferably the agent is calcium chloride.

The mixing with further calcium chloride leads to the formation ofhighly cross-linked plasma-based films which are resistant todegradation in 8M urea over days, to weeks, to months. As such, if theplasma-based film to be prepared is to be a more rapidly degrading film,calcium chloride should not be added. In the absence of calcium chloridethe fibrin in the plasma-based film has a very low degree ofcross-linking and is rapidly degradable. For example, a film with a lowdegree of cross-linking dissolves in 8M urea within about 4 hours.Notwithstanding the above, even if having a high degree ofcross-linking, the films of the present invention are fullybiodegradable.

The blood plasma suitable for use in the methods of the first and secondaspect can be animal or human blood plasma. Typically the plasma isFresh Frozen Plasma (FFP) or is pathogen-safe plasma. In methods wherepathogen-safe plasma is used, the plasma has been subjected to viralinactivation treatment, preferably solvent/detergent treatment (S/Dtreatment), and/or has been pasteurized, and/or has been radiated,and/or has been nano-filtered.

In some embodiments, the plasma-based film of the present invention hasa water content of about 3 to 6% by weight, or of about 4 to 5% byweight, or of about 4% by weight. Residual water content was determinedby evaluation of the weight of the film prior to determination (totalweight) and after drying in an oven at 105° C. to constant weight. Thedifference in weight represents the weight of residual water (prior tooven drying) and is given as % residual water, based on total weightprior to oven drying. Films of the present invention having a residualwater content of about 4% are referred to as “dry” films in the Examplesbelow, while such films after soaking for 10 minutes in an aqueoussolution containing 10% glycerol are referred to as “soaked films”.

The methods of the first and second aspects are suitable for makingflexible, plasma-based films having a thickness ranging from about 0.005to 0.1 mm, or from about 0.005 to 0.09 mm, or from about 0.0075 to 0.08mm, or from about 0.01 to 0.08 mm, or from about 0.01 to 0.07 mm, orfrom about 0.015 to 0.065 mm, or from about 0.02 to 0.06 mm, or fromabout 0.02 to 0.055 mm, or from about 0.02 to 0.05 mm, or from about0.02 to 0.04 mm, or from about 0.02 to 0.03 mm, or about 0.01 mm, orabout 0.02 mm, or about 0.03 mm, or about 0.04 mm, or about 0.05 mm, orabout 0.06 mm. Notwithstanding, films with other thicknesses are alsoderivable by simply up- or down-scaling the volumes of the componentsused in the methods of the first and second aspects.

Further, the thickness and strength of the film correspond to the amountof pressure placed on the top plate and the amount of plasma applied.Films prepared in a 4″×4″ mold pressurized for 30 to 120 seconds with700 pounds force, i.e. about 44 psi or 300 kPa, have a thickness ofabout 0.02 mm when prepared from 30 ml of clotting mixture (such filmsare referred to as “SUBC 30” below). Films prepared correspondingly butfrom 60 ml clotting mixture have a thickness of about 0.03 mm (suchfilms are referred to as “SUBC 60” below) and films preparedcorrespondingly but from 120 ml clotting mixture have a thickness of0.05-0.06 mm (such films are referred to as “SUBC 120” below).

As already described above, the films of the present invention, i.e. thefilms prepared by the methods of the first and second aspects arecharacterized by an advantageous flexibility and, therefore, by: a foldnumber of at least 1, such as at least 2, such as at least 3, such as atleast 4, such as at least 5; a fold endurance of at least 10, such as atleast 20, such as at least 30, such as at least 40, such as at least 50,such as at least 60, such as at least 70, such is at least 80, such asat least 90, such is at least 100; and/or a burst pressure of about 50to 1000 mm Hg, or of about 100 to 1000 mm Hg, or of about 100 to 800 mmHg, or of about 100 to 600 mm Hg, or of about 100 to 500 mm Hg, or ofabout 100 to 450 mm Hg, or of about 140 mm Hg, or of about 150 mm Hg, orof about 175 mm Hg, or of about 200 mm Hg, or of about 225 mm Hg, or ofabout 250 mm Hg, or of about 275 mm Hg, or of about 300 mm Hg, or ofabout 325 mm Hg, or of about 350 mm Hg, or of about 375 mm Hg, or ofabout 400 mm Hg.

In a further preferred embodiment the flexible plasma-based films arecharacterized by a reduced burst pressure after having been soaked in anaqueous solution containing 10% glycerol for 10 minutes. In particular,such soaked films are characterized by a burst pressure of 75% to 80% ofthat of a corresponding “dry” film, i.e. a film having a residual watercontent of about 4% and having the same thickness when dry.

In a further preferred embodiment, the flexible plasma-based film ischaracterized by a ratio of burst pressure/film thickness of 5000 to9000 mm Hg/mm film thickness when dry (i.e. when having a residual watercontent of about 4%) and having a film thickness of 0.05-0.06 mm, or bya ratio of burst pressure/film thickness of 6000 to 7500 mm Hg/mm filmthickness when dry (i.e. when having a residual water content of about4%) and having a film thickness of 0.03 mm.

Accordingly and as already indicated above, the flexible plasma-basedfilms of the present invention are prepared by the methods of the firstand second aspects. Notwithstanding, the flexible plasma-based films ofthe present invention may also be characterized by their specificfeatures already outlined above.

As such, in a fourth aspect the present invention relates to a flexibleplasma-based film comprising between 0.1 to 10 IU of thrombin per ml ofplasma and having a thickness ranging from about 0.005 to 0.1 mm,wherein the flexible film is characterized by a fold number of at least1, such as at least 2, such as at least 4, such as at least 5.Preferably, the flexible plasma-based film is characterized by a foldendurance of at least 10, such as at least 20, such as at least 30, suchas at least 40, such as at least 50, such as at least 60, such as atleast 70, such is at least 80, such as at least 90, such is at least100, and/or by a burst pressure of about 50 to 1000 mm Hg, or of about100 to 1000 mm Hg, or of about 100 to 800 mm Hg, or of about 100 to 600mm Hg, or of about 100 to 500 mm Hg, or of about 100 to 450 mm Hg, or ofabout 140 mm Hg, or of about 150 mm Hg, or of about 175 mm Hg, or ofabout 200 mm Hg, or of about 225 mm Hg, or of about 250 mm Hg, or ofabout 275 mm Hg, or of about 300 mm Hg, or of about 325 mm Hg, or ofabout 350 mm Hg, or of about 375 mm Hg, or of about 400 mm Hg.

In particular, the flexible plasma-based films of the present inventionare suitable for use as a hemostat, preferably as a hemostat to stop amild to severe bleeding, and/or to stop an arterial bleeding. Preferablythe films are also suitable: for use as an anti-adhesive sheet to reduceor prevent development of a surgery-induced adhesion; for use as a woundhealing patch; for use as a wound dressing; or for use in hernia repair.

EXAMPLES

The invention is further described by the following non-limitingExamples.

SUBC Films

SUBC 30

A highly flexible plasma based film was prepared by mixing 30 ml plasmawith either 280-320 μl thrombin (300 IU/ml) or 280-320 μl calciumsolution (2 M Ca²⁺) or 280-320 μl thrombin (300 IU/ml) together withabout 280-320 μl calcium solution (2 M Ca²⁺), subsequently incubatingthe mixture at 25-37° C. for about 15 minutes, in a 4″×4″ mold andpressurizing the obtained plasma clot in the mold with a pressure ofabout 44 psi for about 60 seconds.

Finally, the obtained film was dried in a laminar hood overnight to aresidual moisture content of about 4%. This film is indicated in Tables3 and 4 below as “SUBC 30 (dry)”.

SUBC 60

A highly flexible plasma based film was prepared by mixing 60 ml plasmawith either 580-620 μl thrombin (300 IU/ml) or 580-620 μl calciumsolution (2 M Ca²⁺) or 580-620 μl thrombin (300 IU/ml) together withabout 580-620 μl calcium solution (2 M Ca²⁺), subsequently incubatingthe mixture at 25-37° C. for about 15 minutes, in a 4″×4″ mold andpressurizing the obtained plasma clot in the mold with a pressure ofabout 44 psi for about 60 seconds.

Finally, the obtained film was dried in a laminar hood overnight to aresidual moisture content of about 4%. This film is indicated in Tables3 and 4 below as “SUBC 60 (dry)”.

Some of the SUBC 60 (dry) films were subsequently soaked in 10% glycerolfor 10 minutes. Such films are indicated as “SU BC 60 (soaked)”.

SUBC 120

A highly flexible plasma based film was prepared by mixing 120 ml plasmawith either 1180-1220 μl thrombin (300 IU/ml) or 1180-1220 μl calciumsolution (2 M Ca²⁺) or 1180-1220 μl thrombin (300 IU/ml) together withabout 1180-1220 μl calcium solution (2 M Ca²⁺), subsequently incubatingthe mixture at 25-37° C. for about 15 minutes, in a 4″×4″ mold andpressurizing the obtained plasma clot in the mold with a pressure ofabout 44 psi for about 60 seconds.

Finally, the obtained film was dried in a laminar hood overnight to aresidual moisture content of about 4%. This film is indicated in Tables3 and 4 below as “SUBC 120 (dry)”.

Some of the SUBC 120 (dry) films were subsequently soaked in 10%glycerol for 10 minutes-. Such films are indicated as “SUBC 120(soaked)”.

CE Films

Comparative Example 1—CE-1

One prior-art blood plasma based film is known from EP 0485210 A2.Production method “4. Plasma Membrane” teaches mixing of 50 ml citratedplasma with 8-10 NIH units thrombin in a mold apparatus at 4° C. andwarming to room temperature or 37° C. Obtained films might eventually bedried overnight in a laminar flow hood, compressed or desiccated. Inorder to get most comparable results the mixture was up-scaled to 60 ml.In particular 60 ml of plasma were mixed with 12 NIH units of thrombin.The mixture was warmed to 37° C. in a 4″×4″ mold and was kept for 2 hourat this temperature. Finally the films were compressed with 44 psi for60 seconds, excess water and serum was removed and the films were driedovernight under a laminar flow hood.

Thus prepared films are indicated in tables 1 and 2 as “CE-1 60 (dry)”and films that were additionally soaked in 10% glycerol for 10 minutesare indicated as “CE-1 60 (soaked)”.

Comparative Example 2—CE-2

U.S. Pat. No. 8,529,959 B2 discloses a sheet comprising a bloodplasma-derived plastic, which is pliant, elastic or a combinationthereof.

The source material (a powder of clotted plasma) was obtained asdescribed in Example 11 by clotting of 52.6 parts plasma by addition of1 part 1M calcium chloride in water and lyophilization of the clot for72 hours at a reduced pressure of 6 mTorr. Plasma powder was achieved bygrinding the dried material in a mechanical grinder then sieving througha 150 μm sieve. Further procedures to formulate and form theplasma-derived plastic were the following. To formulate the plastic, 650mg of plasma powder and 350 mg of glycerol (as plasticizer) were addedto a small beaker. The components were mixed until homogeneous andallowed to incubate at room temperature in a closed container forapproximately 21 hours. The resulting “dough” was molded in a press at59° C. An actual description of forming a sheet or film is not given,but U.S. Pat. No. 8,529,959 B2 refers to U.S. patent application Ser.No. 11/495,115 (U.S. Ser. No. 11/495,115) to process elastic sheets.

Said patent application unfortunately only contains information aboutproduction of fibrinogen and gelatin based films. By combination ofteachings disclosed in Examples 24, 16, 2 and 1 the common process ofmixing a powder (ground fibrin, ground plasma clot, ground gelatin ornon-polymerized fibrinogen) with various amounts of a plasticizer(glycerol) to produce a “dough” and forming the dough in a heated presswas deducible. It was also indicated that films prepared from a doughcontaining 12.5% glycerol were most flexible and that films could bemade at any pressure with 1000 to 8000 lbs.

It was thus decided to undertake experiments with non-polymerizedfibrinogen and a plasma clot powder prepared as taught in U.S. Pat. No.8,529,959 B2, admixing 12.5% of glycerol as taught in U.S. Ser. No.11/495,115 or 35% as taught in U.S. Pat. No. 8,529,959 B2 and finalizethe production process by pressure molding in a down-scaled 2″×1.6″ moldat 59° C. and 5000 pounds. Initial experiments with doughs containing35% glycerol revealed that obtained films were quite rigid, hardlyflexible and it was not possible to achieve a single fold with such afilm. Films prepared with 12.5% glycerol were to some extent flexibleand this approach was examined further. Films prepared with 12.5% water,which is also mentioned in the patent (application) as plasticizer, butno glycerol revealed practically no cohesion.

Based on the initial experience of rather poor film properties it wasdecided to produce films comparable in plasma content to 120 ml films ofthe present invention. In order to get most comparable results aportion, being equivalent to the area of the 4″×4″ mold divided by thearea of the down-scaled mold, of 120 ml plasma was clotted andlyophilized as taught in U.S. Pat. No. 8,529,959 B2. The weight of thisclot, i.e. 88-92 mg, in particular about 90 mg, had been determined andserved as the weight of powderized plasma clot to be used for theexperiments resembling a 120 ml film. The films were to some degreepliable, flexible and almost managed to survive one folding in the foldnumber test, but finally they failed to reach a fold number of 1. It waspossible to determine burst pressure, but the results of dry films wereso poor that they were not tested as soaked films. Comparative filmsresembling a 120 ml film are indicated in tables 1 and 2 as “CE-2 120(dry)”.

Comparative Example 3 (CE-3)

CE-3 is a commercially available anti-adhesion film used in theanti-adhesion animal study.

Comparative Example 4 (CE-4)

CE-4 is a commercially available anti-adhesion film used in theanti-adhesion animal study.

Anti-Adhesive Films 1 to 4

Film 1

Initially a film in accordance with the preparation of the SUBC 120(dry) film described above was prepared.

Subsequently the film was soaked in an anti-adhesive agent solutionprior to use. In particular, the SUBC 120 (dry) film was soaked in 7.5ml of a solution consisting of 0.5 ml Tween-80®, 0.25 ml glycerin, 15 mgheparin, and 6.75 ml water. Thus soaked Tween-80® films were used in ananti-adhesion animal study and the results are given in Table 2indicated as “Film 1”.

Film 2

Initially a film in accordance with the preparation of the SUBC 120(dry) film described above was prepared.

However, during preparation of the SUBC 120 (dry) film 1-2% of ananti-adhesive agent, such as Pluronic F68®, was admixed with the plasma.This film can either be used as dry film or as a soaked film aftersoaking. Soaking of a Pluronic F68® containing 120 ml plasma film in 7.5ml of a solution consisting of 0.75 ml glycerin, 15 mg heparin, and 6.75ml water provided a good anti-adhesion barrier.

Thus soaked pluronic films were used in an anti-adhesion animal studyand the results are given in Table 2 indicated as “Film 2”.

Film 3

Initially a film in accordance with the preparation of the SUBC 120(dry) film described above was prepared.

However, during preparation of the SUBC 120 (dry) film 1-2% of twoanti-adhesive agents at equal weight were admixed with the plasma.Namely, equal amounts of Tween-80® and Pluronic F68® by weight wereadmixed with the plasma

Such films were used in an anti-adhesion animal study and the resultsare given in Table 2 indicated as “Film 3”.

Film 4

Initially a film in accordance with the preparation of the SUBC 120(dry) film described above was prepared.

Subsequently, anti-adhesive agents were topically applied to the film.Namely, a COX-2 inhibitor, such as Nimesulide® was applied in 1 mg/cm²by spray-drying, and the film was used as a dry film.

Such films with Nimesulide® being topically applied in 1 mg/cm² wereused in an anti-adhesion animal study and the results are given in Table2 indicated as “Film 4”.

Example 1

Hemostatic Films—Animal Study for Determination of Efficacy.

Highly flexible plasma-based films for use as hemostats in accordancewith the preparation of the SUBC 120 (dry) film described above wereprepared.

The films were then subsequently modified by topical application forfast hemostasis with thrombin. Similar films were also prepared withfibrinogen being spray dried on one side of a film, followed by coveringthe fibrinogen with a layer of starch and finally spray-drying thrombinon top of the starch.

Efficacy of hemostatic films was evaluated in a porcine animal model byinducing a spleen, kidney or liver injury using a template bleedingdevice, which created a shallow wound of approximately 1 cm in diameter.

Evaluation of efficacy was performed after manually pressing the film onthe wound for 3 minutes and waiting for 15 minutes thereafter withoutpressing. The flexible plasma-based films of the present inventiontested had a hemostatic pharmaceutically-active agent topically applied.Namely, hemostats comprising topically applied thrombin, activatedcoagulation factor VII (FVIIa) or a combination of thrombin andfibrinogen were tested.

Hemostatic films with thrombin or a combination of thrombin andfibrinogen were most effective and stopped bleedings within 3 minutes.Films with FVIIa were slightly less effective as a particularly strongarterial bleeding could not be completely stopped by one FVIIa filmafter several minutes, in order to stop this bleeding a thrombin coatedfilm of the present invention, which stopped this arterial bleeding, wasfinally used.

Example 2

Anti-Adhesive Sheets—Animal Study for Determination of Efficacy.

Efficacy of anti-adhesive sheets according to the present invention wasevaluated in a rat animal model. In each animal, standardized surgicalinjuries (2.0×2.5 cm area) were applied to both the right and leftsidewall peritoneum and uterine horns using a cytobrush until punctuatebleeding occurred.

The assigned test or control sheets were applied to cover the abrasionon each uterine horn and sutured into place. Control films werecommercially available implants with dedicated anti-adhesive function.At day 7 following surgery, all animals were humanely euthanized andsubjected to necropsy for gross assessment of adhesions.

Quality and Quantity of Adhesions were Evaluated as Follows:

Quantity: The quantity was scored according to the incidence oftraumatized areas with adhesions and the adhesion coverage (which iscalculated as a fraction of adhesions and translated into a percentageof traumatized area).

Quality: The quality of the induced adhesions was scored in accordancewith Table 1. The results of quality and quantity are presented in Table2. Adhesion quality was considered “filmy” if the scale of a ruler wasvisible through the tissue, and otherwise considered “dense.”

TABLE 1 (Scoring of Adhesion Quality) Score Description 0 No adhesion 1A vascular adhesion 2 Filmy vascular adhesion 3 Dense, vascularadhesion, uterine horn not visible due to adhesiogenesis

TABLE 2 (Quality and Quantity of anti-adhesion results) Quantity [%Surface Anti-adhesion area Covered with film Quality Adhesions] Film 1 0 0 Film 2 1 10-15  Film 3 0-1 0-15 Film 4 0-1 0-10 CE-3 2-3 70-100 CE-42 40

Films 1-4 of the present invention performed remarkably better thancommercially available anti-adhesion films.

Example 3

Assessment of Film Flexibility.

The flexibility of the plasma-based films of the present invention,namely for films SUBC 30 (dry), SUBC 60 (dry) and (soaked), SUBC 120(dry) and (soaked) and for the comparative films CE-1 60 (dry) and(soaked) and CE-2 120 (dry) was assessed by determining the fold number,fold endurance and burst pressures as described above.

Results

TABLE 3 (Fold Number, Fold Endurance) Film - ml Fold Nr. Fold EnduranceSUBC 30 (dry) 5 100 SUBC 60 (dry) 5 100 SUBC 60 5 100 (soaked) SUBC 120(dry) 5 100 SUBC 120 5 100 (soaked) CE-1 60 (dry) 0 0 CE-1 60 (soaked) 00 CE-2 120 (dry) 0 0

As none of comparative films CE-1 and CE-2 managed to achieve a singlefold without breaking it was not possible to determine fold endurance.

TABLE 4 (Burst Pressure) Burst average Thickness Pressure Burst Pr.Burst Pr./mm Film - ml [mm] [mmHg] [mm Hg] Thickness SUBC 30 (dry) 0.02SUBC 60 (dry) 0.03 186-218 198 6200-7267 SUBC 60 (soaked) 149-162 156SUBC 120 (dry) 0.05-0.06 318-441 404 5300-8820 SUBC 120 (soaked) 194-400314 CE-1 60 (dry) 0 CE-1 60 (soaked) 10-33 CE-2 120 (dry) 20-25 CE-1(dry) films were so brittle and inflexible that they could not be placedin the fixture without breaking them. It was thus not possible todetermine burst pressure for these films.

Many modifications and other embodiments of the invention set forthherein will come to mind to the one skilled in the art to which theinvention pertains having the benefit of the teachings presented in theforegoing description and the associated drawings. Therefore, it is tobe understood that the invention is not to be limited to the specificembodiments disclosed and that modifications and other embodiments areintended to be included within the scope of the appended claims.Although specific terms are employed herein, they are used in a genericand descriptive sense only and not for purposes of limitation.

LIST OF REFERENCE SIGNS

-   -   1 plasma-based film    -   2 pharmaceutically-active agent    -   3 further pharmaceutically-active agent-spray-dried plasma    -   4 further pharmaceutically-active agent-spray-dried activator of        the coagulation system    -   5 clotting mixture    -   6 solid, non-porous plate    -   7 mold    -   8 liquid removal system, such as vacuum-driven suction    -   9 downward piston    -   10 absorbent material

What is claimed is:
 1. A flexible plasma-based film comprising between2.5 and 10 International Units (IU) of thrombin per ml of undilutedplasma and having a thickness ranging from about 0.005 to 0.1 mm,wherein said flexible film is characterized by a burst pressure of about50 to 1000 mm Hg obtainable by subjecting the film to a pressure of upto 125 psi in a mold.
 2. The flexible plasma-based film according toclaim 1, wherein said flexible film is characterized by a burst pressureof about 100 to 1000 mm Hg.
 3. The flexible plasma-based film accordingto claim 1, wherein said flexible film is characterized by a watercontent of about 3 to 6% by weight.
 4. The flexible plasma-based filmaccording to claim 1, wherein said flexible film comprises at least onepharmaceutically-active agent.
 5. The flexible plasma-based filmaccording to claim 1, wherein said flexible film comprises at least onepharmaceutically-active agent homogeneously distributed throughout saidfilm.
 6. The flexible plasma-based film according to claim 4, whereinthe at least one pharmaceutically-active agent is selected from thegroup consisting of: antibiotic agents; anti-inflammatory agents;anti-infective agents; growth factors; chemokines; immunomodulators;wound healing agents; activators of the coagulation system;anti-coagulation agents; anti-adhesion agents; anti-fibrinolytic agents;penicillin; silver; chlorhexidine; stromal cell-derived chemokines;stromal cell-derived factor 1 alpha; stromal cell-derived factor 1 beta;fibrinogen; Factor VIIa; CXCL-12; heparin; aprotinin; tranexamic acid;non-ionic surfactants; polyoxyethylene-polyoxypropylene block copolymer;polysorbate 80; COX-2 inhibitors; and Nimesulide.
 7. The flexibleplasma-based film according to claim 5, wherein the at least onepharmaceutically-active agent is selected from the group consisting of:antibiotic agents; anti-inflammatory agents; anti-infective agents;growth factors; chemokines; immunomodulators; wound healing agents;activators of the coagulation system; anti-coagulation agents;anti-adhesion agents; anti-fibrinolytic agents; penicillin; silver;chlorhexidine; stromal cell-derived chemokines; stromal cell-derivedfactor 1 alpha; stromal cell-derived factor 1 beta; fibrinogen; FactorVIIa; CXCL-12; heparin; aprotinin; tranexamic acid; non-ionicsurfactants; polyoxyethylene-polyoxypropylene block copolymer;polysorbate 80; COX-2 inhibitors; and Nimesulide.
 8. The flexibleplasma-based film according to claim 1, wherein said plasma is animal orhuman blood plasma.
 9. The flexible plasma-based film according to claim1, wherein said plasma is Fresh Frozen Plasma (FFP) or is pathogen-safeplasma.
 10. The flexible plasma-based film according to claim 1, whereinsaid film comprises one or more humectants.
 11. The flexibleplasma-based film according to claim 1, wherein said film comprises anadhesive backing.
 12. The flexible plasma-based film according to claim1, wherein said film is a sterilized film.
 13. The flexible plasma-basedfilm according to claim 1, wherein said film is packaged in ahumidity-controlled container.
 14. The flexible plasma-based filmaccording to claim 1, wherein said flexible film is characterized by aburst pressure of about 100 to 800 mm Hg.
 15. The flexible plasma-basedfilm according to claim 1, wherein said flexible film is characterizedby a burst pressure of about 100 to 600 mm Hg.